1. Field of the Invention
The present invention relates to improved methods for the incorporation of DNA into the genome of a Zea mays plant by means of Agrobacterium-mediated transformation.
2. Description of the Related Art
During the past decade, it has become possible to transfer genes from a wide range of organisms to crop plants by recombinant DNA technology. This advance has provided enormous opportunities to improve plant resistance to pests, diseases and herbicides, and to modify biosynthetic processes to change the quality of plant products. However, the availability of an efficient transformation method to introduce foreign DNA remains to be a substantial barrier for most monocot species, including maize.
There have been many methods attempted for the transformation of monocotyledonous plants, wherein “biolistics” is the most widely used transformation method. In the “biolistics” (microprojectile-mediated DNA delivery) method microprojectile particles are coated with DNA and accelerated by a mechanical device to a speed high enough to penetrate the plant cell wall and nucleus (WO 91/02071). The foreign DNA gets incorporated into the host DNA and results in a transformed cell. There are many variations on the “biolistics” method (Sanford 1990; Fromm 1990; Christou 1988; Sautter 1991). The method has been used to produce stably transformed monocotyledonous plants including rice and maize (Christou 1991; Gordon-Kamm 1990; Vasil 1992, 1993; Wan 1994; Sommers 1992). However, even with the more recent improvements it still requires 4 to 6 months to recover transgenic plants (Weeks 1993; Vasil 1992, 1993; Becker 1994, Rasco-Gaunt 2001). Microprojectile-mediated DNA delivery brings about a number of problems such as frequent fragmentation of the DNA prior to its integration, random integration in transcribed as well as non-transcribed chromosomal regions, predominantly multiple insertion of the sequence to be transferred, complex integration patterns, integration of backbone sequences including selectable marker genes at the same locus. Moreover, microprojectile-mediated plant transformation is generally based upon genotype-dependent cell culture methods which often require a secondary transfer of the transgene into the background of elite breeding material via long-lasting back-crossing.
Protoplast based methods have been used mostly in rice, where DNA is delivered to the protoplasts through liposomes, PEG, or electroporation (Shimamoto 1989; Datta 1990b). Protoplasts may be isolated from various tissues but require in general the use of cell wall-degrading enzymes. It is considered likely that the use of cell wall-degrading enzymes can inhibit the subsequent regeneration process. Furthermore, most protoplast based methods require the establishment of long-term embryogenic suspension cultures. Some regenerants from protoplasts are infertile and phenotypically abnormal due to somaclonal variation during the long-term suspension culture (Davey 1991; Rhodes 1988). Transformation by electroporation involves the application of short, high-voltage electric fields to create “pores” in the cell membrane through which DNA is taken-up. This method has been used to produce stably transformed monocotyledonous plants (Paszkowski 1984; Shillito 1985; Fromm 1986) especially from rice (Shimamoto 1989; Datta 1990b; Hayakawa 1992).
A number of other methods have been reported for the transformation of monocotyledonous plants including, for example, the “pollen tube method” (WO 93/18168; Luo 1988), macro-injection of DNA into floral tillers (Du 1989; De la Pena 1987), injection of Agrobacterium into developing caryopses (WO 00/63398), and tissue incubation of seeds in DNA solutions (Töpfer 1989). Direct injection of exogenous DNA into the fertilized plant ovule at the onset of embryogenesis was disclosed in WO 94/00583.
While widely useful in dicotyledonous plants, Agrobacterium-mediated gene transfer has long been disappointing when adapted to use in monocots. There are several reports in the literature claiming Agrobacterium transformation of monocotyledons (e.g., discussed WO 94/00977). These are specifically the methods of Gould 1991; Mooney 1991; and Raineri 1990, which claim Agrobacterium transformation of maize, rice and wheat. There is some evidence of gene transfer in these methods but they lack convincing evidence for transfer efficiency, reproducibility, and confirmation of gene transfer (Potrykus 1990), and lack of evidence of the transgene inheritance in the progeny when plants are produced. In the work of Gould where evidence of transformed plants was presented there was no Mendelian inheritance of the genes. Attempts by Hiei et al. (1994) suggested that transgenic rice plants could be obtained following Agrobacterium-mediated transformation, but the particular bacterial strains used and the choice of bacterial vectors were critical for successfully obtaining transgenics. A paper by Ishida et al. (1996) indicated that high-efficiency transformation of maize was possible by co-culture of immature embryos with A. tumefaciens. In both reports on rice and maize transformation, a super-binary vector pTOK233 containing additional copies of the virB, virC and virG genes was used to achieve high-efficiency transformation. WO 95/06722 and EP-A1 672 752 disclose a method of transforming monocotyledons using scutellum of immature embryos with A. tumefaciens, which immature embryo has not been subjected to a dedifferentiation treatment. EP-A1 0 709 462 describes a method for transforming monocotyledonous plants, wherein the improvement is pointed out to include a recovery period after the co-cultivation step without a selection device for one day.
Although the methods known in the art, especially those provided in WO 95/06722, provide means for producing transgenic Zea mays plants, all these methods are still time and labor intensive. Especially when large numbers of transgenic plants need to be established (such as for example in a genomics based screening approach requiring construction of hundreds to thousands of different transgenic plants) efficiency is of high commercial importance.
Accordingly, the object of the present invention is to provide an improved, efficient method for transforming Zea mays plants, with which the time required for obtaining regenerated plants from the time of transformation is shorter than that in the conventional methods. This objective is achieved by the present invention.